Structural and regulatory studies on protein components of the Escherichia coli (E. coli) sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. The phosphorylated form of the amino terminal domain (EIN) of the first enzyme (EI) of the pathway was characterized. NMR studies showed that the active site Histidine 189 is phosphorylated at the N-epsilon-2 position and has a pKa of 7.3 (one pH unit higher than that of unphosphorylated Histidine 189). Results indicate that both protonation and phosphorylation of Histidine 189 must be accompanied by a change in the side-chain conformation of Histidine 189. Thermal stability studies, using differential scanning calorimetry (DSC) and far-UV circular dichroism (CD), showed that phospho-EIN and phospho-EI are destabilized compared to the unphosphorylated forms of the proteins. This decrease in conformational stability was associated with promotion of phosphoryl transfer from EI to its acceptor protein, HPr. Ligand fishing experiments, using surface plasmon resonance, revealed that E. coli HPr exhibits a high affinity interaction with glycogen phosphorylase (GP). HPr allosterically regulates the oligomeric state of GP. Binding of HPr increases GP activity by decreasing the Km for glycogen and increasing the Vmax for phosphate. The unphosphorylated form of HPr activates GP more than does the phosphorylated form, suggesting a regulatory mechanism controlled by the state of phosphorylation of HPr. In Gram-positive organisms, HPr can be phosphorylated on Serine 46 by a specific ATP-dependent kinase. This phosphorylation plays a role in controlling catabolite repression. The specificity requirements for phosphorylation of Mycoplasma capricolum HPr were determined by mutagenesis of HPr. Residues 48, 49, and 51-53 were found to be important for recognition of Mycoplasma HPr by its cognate HPr(ser) kinase. The characteristics of this region suggest that the kinase-HPr interaction occurs mainly through a hydrophobic region. In the PTS pathway, phosphorylated HPr transfers its phosphoryl group to IIAglc, The crystal structure of Mycoplasma capricolum IIAglc was solved. Two neighboring IIAglc molecules associate with one another in a front-to-back fashion, such that Glutamic acid 149 of one molecule forms electrostatic interactions with the active site histidine residues, Histidine 90 and Histidine 75, of the other. Glutamate 149 is therefore considered to mimic the interaction that a phosphorylated histidine of a partner protein makes with IIAglc. The non-discriminatory nature of the hydrophobic interactions that IIAglc forms with a variety of partners may be a consequence of the requirement for interaction with a variety of proteins that show no sequence or structural similarity. Nevetheless, specificity is provided by an ion-pair interaction that is enhanced by the apolar nature of the interface. IIAglc of E. coli allosterically regulates the activity of lactose permease (LP). The nature of the interaction of IIAglc with the cytoplasmic face of LP was explored. LP mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind IIAglc, indicating that these regions of LP may be involved in recognition of IIAglc. The results suggest that interaction of LP with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with IIAglc. A topological map of the proposed interaction was formulated.